CN113073080A - Polymorphic rhabdomyosarcoma organoid and culture method, culture medium and application thereof - Google Patents
Polymorphic rhabdomyosarcoma organoid and culture method, culture medium and application thereof Download PDFInfo
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Abstract
The present disclosure relates to a polymorphic rhabdomyosarcoma organoid and its culture method, culture medium and use, the culture medium contains 740Y-P25-75 μ g/mL, MHY1485 5-15 μ M, mitotic growth factor 80-120ng/mL, Wnt agonist 90-110ng/mL, ROCK inhibitor 4-15 μ M, BMP inhibitor 10-15ng/mL, 10-15 μ M of a p38 kinase inhibitor, 0.5-5 vol.% N2 based on the total volume of the medium, 0.5-5 vol.% B27 based on the total volume of the medium, 1-5mM HEPES, 2-5mM Glutamax, 2-5mM acetylcysteine, 150-. The culture medium disclosed by the invention can be used for three-dimensional culture of the polymorphic rhabdomyosarcoma organoids, and the number of the cultured tumor organoids is large and the survival rate is high.
Description
Technical Field
The disclosure relates to the technical field of biomedicine, in particular to a polymorphic rhabdomyosarcoma organoid and a culture method, a culture medium and application thereof.
Background
Rhabdomyosarcoma (RMS) is a soft tissue malignancy consisting of rhabdomyoblasts of various degrees of differentiation, with a rate of incidence second to that of malignant fibrous histiocytoma and liposarcoma, the third place in soft tissue sarcomas. The WHO histologically classified RMS into three subtypes: embryonal Rhabdomyosarcoma (ERMS), Acinar Rhabdomyosarcoma (ARMS), and rhabdomyosarcoma of Pleomorphy (PRMS). Embryonal rhabdomyosarcoma accounts for about 2/3 of rhabdomyosarcoma, and occurs well in children and adolescents, and the age distribution shows two peaks, namely, after birth and later stage of adolescence, and the average age is 5 years old. Alveolar rhabdomyosarcoma is found in adolescents, more male than female. Rhabdomyosarcoma of multiforme occurs mainly in adults, mostly in the age of 40-70 years.
The pathogenesis of RMS is not clear at present, different subtypes of RMS can have completely different pathogenesis, individual differences are obvious, and polymorphic rhabdomyosarcoma is a very rare tumor without exact pathogenesis, and the diagnosis is more difficult.
Research shows that tumor organoids can be used as a drug screening model to be applied to drug effectiveness detection so as to assist in formulating personalized diagnosis and treatment schemes. However, the culture method for the polymorphic rhabdomyosarcoma organs is not complete, the difference between the in vitro culture environment and the in vivo culture environment causes cell death, the culture conditions for maintaining the in vitro growth and passage of the cells are not clear, and the problems of small culture quantity of the tumor organs, low survival rate and the like exist.
Disclosure of Invention
The invention aims to provide a rhabdomyosarcoma organoid with pleomorphism, a culture method, a culture medium and application thereof.
To achieve the above objects, the first aspect of the present disclosure provides a culture medium for culturing rhabdomyosarcoma organoids of multiforme, said medium comprising 25-75 μ g/mL 740Y-P, 5-15 μ M MHY1485, 80-120ng/mL mitotic growth factor, 90-110ng/mL Wnt agonist, 4-15 μ M ROCK inhibitor, 10-15ng/mL BMP inhibitor, 10-15 μ M P38 kinase inhibitor, optionally 0.5-5% by volume N2 based on the total volume of said medium, optionally 0.5-5% by volume B27 based on the total volume of said medium, optionally 1-5mM HEPES, optionally 2-5mM Glutamax, optionally 2-5mM acetylcysteine, optionally 150U/mL penicillin-streptomycin, and optionally 150U/mL penicillin-streptomycin based on the total volume of said medium Volume based 5-15% by volume of FBS.
In a second aspect, the present disclosure provides a method of culturing a rhabdomyosarcoma organoid of pleomorphism, the method comprising:
s1, mixing normal tissue cells beside the pleomorphic rhabdomyosarcoma cancer, a culture medium and matrigel, adding the mixture into the holes of the culture plate, and carrying out first culture to obtain a collagen layer of the normal tissue beside the cancer;
s2, mixing the pleomorphic rhabdomyosarcoma cells, the matrigel and the laminin, then coating the mixture on the collagen layer of the paracancer normal tissue, and carrying out second culture to obtain a double-layer matrix culture system;
s3, adding the culture medium provided by the first aspect of the disclosure into the double-layer matrix culture system for amplification culture.
In a third aspect of the present disclosure, there is provided a polymorphic rhabdomyosarcoma organoid cultured by the method provided in the second aspect of the present disclosure.
The fourth aspect of the present disclosure provides a use of the rhabdomyosarcoma organoid of polymorphism provided by the third aspect of the present disclosure in drug screening and tumor mechanism research.
Through the technical scheme, the culture medium disclosed by the invention contains 740Y-P and MHY1485, and the pleomorphic rhabdomyosarcoma cells and the paracancer normal tissue cells are subjected to three-dimensional co-culture by adopting the culture medium, so that the growth of the pleomorphic rhabdomyosarcoma cells can be effectively promoted, and the cultured pleomorphic rhabdomyosarcoma organs are large in number and high in survival rate. Polymorphic rhabdomyosarcoma organs obtained by culture are adopted to carry out drug screening, and personalized diagnosis and treatment schemes can be formulated for different patient individuals.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
In a first aspect of the present disclosure, there is provided a culture medium for culturing a rhabdomyosarcoma organoid of multiforme, said medium comprising 740Y-P at 25-75 μ g/mL, MHY1485 at 5-15 μ M, mitotic growth factor at 80-120ng/mL, Wnt agonist at 90-110ng/mL, ROCK inhibitor at 4-15 μ M, BMP inhibitor at 10-15ng/mL, P38 kinase inhibitor at 10-15 μ M, N2 at 0.5-5% by volume based on the total volume of said medium, B27 at 0.5-5% by volume based on the total volume of said medium, HEPES at 1-5mM, Glutamax at 2-5mM, acetylcysteine at 2-5mM, penicillin-streptomycin at 150U/mL, and penicillin-streptomycin at 5- 15% by volume of FBS.
The inventors of the present disclosure unexpectedly found that a culture medium containing 740Y-P and MHY1485 is effective in promoting the growth of rhabdomyosarcoma cells of pleomorphy, and that the culture medium, when used for three-dimensional culture of rhabdomyosarcoma cells of pleomorphy and normal tissue cells beside cancer thereof, can significantly improve the culture number and survival rate of tumor-like organs.
In a preferred embodiment of the invention, 740Y-P is present in the medium in an amount of 30-70. mu.g/mL and MHY1485 is present in an amount of 6-13. mu.M. More preferably, 740Y-P is present in an amount of 40-60. mu.g/mL and MHY1485 is present in an amount of 8-11. mu.M.
In a preferred embodiment of the present disclosure, the medium further comprises 90-110ng/mL mitotic growth factor, 92-105ng/mL Wnt agonist, 5-14 μ M ROCK inhibitor, 12-14ng/mL BMP inhibitor, 11-14 μ M p38 kinase inhibitor, optionally 1-4% by volume N2 based on the total volume of the medium, optionally 1-2% by volume B27 based on the total volume of the medium, optionally 2-4mM HEPES, optionally 3-5mM Glutamax, optionally 3-4mM acetylcysteine, optionally 170-. Wherein, each component of the culture medium can be obtained by purchase, and the composition, the formulation, the specification and the like of each component are known in the field and are not described in detail herein. In the embodiment of the disclosure, the culture medium obtained by reasonably matching the components is used for three-dimensional culture of the rhabdomyosarcoma cells and the paracancer normal tissue cells thereof, so that more rhabdomyosarcoma organs with multiple polymorphisms can be obtained through culture, and the survival rate of the organs is higher.
Mitotic growth factors, Wnt agonists, ROCK inhibitors, BMP inhibitors, and p38 kinase inhibitors are all well known to those skilled in the art in light of this disclosure. Preferably, the mitotic growth factor may be EGF and/or FGF; the Wnt agonist can be one or more selected from Wnt, Wnt-3a and R-Spondin-4; the ROCK inhibitor can be A8301 and/or Y-27632; the BMP inhibitor may be selected from Noggin and/or LDN-212854; the p38 kinase inhibitor can be one or more selected from SB202190, Dilmapimod and SKF-86002.
In a second aspect, the present disclosure provides a method of culturing a rhabdomyosarcoma organoid of pleomorphism, the method comprising: s1, mixing normal tissue cells beside the pleomorphic rhabdomyosarcoma cancer, a culture medium and matrigel, adding the mixture into the holes of the culture plate, and carrying out first culture to obtain a collagen layer of the normal tissue beside the cancer; s2, mixing the pleomorphic rhabdomyosarcoma cells, the matrigel and the laminin, then coating the mixture on the collagen layer of the paracancer normal tissue, and carrying out second culture to obtain a double-layer matrix culture system; s3, adding the culture medium provided by the first aspect of the disclosure into a double-layer matrix culture system for amplification culture.
In the method disclosed by the invention, the pleomorphic rhabdomyosarcoma cells and the paracancer normal tissue cells thereof are cultured in a three-dimensional way by using a culture medium containing 740Y-P and MHY1485, so that the cultured pleomorphic rhabdomyosarcoma organs are large in number and high in survival rate.
In a preferred embodiment of the present disclosure, in step S1, the pleomorphic rhabdomyosarcoma paranormal tissue cells may be placed in culture wells of a culture plate (which may be a 96-well culture plate, for example) such that the primary culture is 500-1500 per well, followed by the first culture. The conditions of the first culture may include: the temperature is 30-40 deg.C, preferably 37 deg.C, and the time is 0.2-2 hr, preferably 0.5 hr. The kind of Matrigel is not particularly limited in the present disclosure, and may be conventionally used by those skilled in the art, and may be, for example, Matrigel. The amount of matrigel may vary within wide limits, for example 10-15. mu.L of matrigel relative to 10. mu.L of the culture medium.
In a preferred embodiment of the present disclosure, in step S2, the step of mixing the rhabdomyosarcoma carcinoma pleomorphic cells, the matrigel, and the laminin, coating the mixture on the collagen layer of the paracarcinoma normal tissue, and performing the second culture comprises: carrying out heavy suspension treatment on the rhabdomyosarcoma carcinoma cells by adopting a culture medium, and mixing a heavy suspension treatment mixture, matrigel and laminin to obtain a first mixture; dripping the first mixture onto the collagen layer of paracancer normal tissue, and performing second culture at 30-40 deg.C for 0.2-2 hr. The concentration of rhabdomyosarcoma carcinoma cells in the resuspension treatment mixture can vary over a wide range, and can be, for example, 20-60 cells/μ L. The amount of laminin may vary within wide limits, for example, the amount of laminin may be 40-60 μ g relative to 1mL of resuspension treatment mixture.
In a preferred embodiment of the present disclosure, in step S3, the adding the culture medium provided in the first aspect of the present disclosure to the two-layer matrix culture system for performing the amplification culture includes: adding 50-150 μ L of culture medium into the double-layer matrix culture system, and performing amplification culture at 30-40 deg.C until each well has 20-40 rhabdomyosarcoma organoids with a diameter of 200-300 μ M. Preferably, the medium is changed at day 3 of the amplification culture (e.g., half of the medium is changed to fresh medium) and then the medium is changed every 2-3 days until there are 20-40 rhabdomyosarcoma organoids of 200-300 μ M diameter in each well.
According to the present disclosure, the method of the present disclosure may further include: mixing the amplification culture product obtained by amplification culture in the step S3 with a cell digestion solution, and performing cell digestion treatment to obtain a second mixture; terminating the cell digestion of the second mixture, and subculturing the cells obtained after the termination of the cell digestion.
The type of the cell digestive juice is not specifically limited in the disclosure, the cell digestive juice can be Tryple Express, Trypsin-EDTA Solution, Acctuase and the like, the dosage of the cell digestive juice can be selected according to actual needs, and preferably, the dosage is 5 × 105The dosage of the cell mass is 2-5mL, and the digestion treatment conditions comprise: the temperature is 30-40 deg.C, and the time is 2-6 min.
According to the present disclosure, the method of allowing the second mixture to terminate the cell digestion is not limited, and for example, DMEM medium having an FBS concentration of 5-15 vol% may be mixed with the second mixture to terminate the cell digestion.
In a third aspect of the present disclosure, there is provided a polymorphic rhabdomyosarcoma organoid cultured by the method provided in the second aspect of the present disclosure.
In a fourth aspect of the present disclosure, there is provided a use of the rhabdomyosarcoma organoid of the polymorphic form provided in the third aspect of the present disclosure in drug screening and tumor mechanism research.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
The raw materials, reagents, instruments and equipment mentioned in the examples of the present disclosure can be purchased without specific description, and when the specific test temperature is not specified in the examples of the present disclosure, the test temperature is room temperature (20 to 25 ℃).
The sources of reagents used in the examples of the present disclosure are as follows:
DMEM medium was purchased from Hyclone, USA; cosmo temperature-sensitive hydrogels were purchased from Cosmo Bio Inc.; fetal bovine serum albumin (FBS) was purchased from inner mongolia jinyuan kang bioengineering ltd; Penicillin-Streptomycin (Penicillin-Streptomycin) was purchased from shanghai bio-engineering gmbh; p160ROCK inhibitor, Y-27632 dihydrochloride inhibitor, collagene type II, R-Spondin, Glutamax, laminin, available from Invitrogen, USA; EGF, Wnt-3a, SB202190, HEPES, available from Sigma- -Aldrich, USA; a8301, MHY1485, 740Y-P are available from MedChemexpress, USA.
The culture medium employed in example 1 of the present disclosure contains: 100ng/mL EGF, 100ng/mL Wnt-3a, 1. mu.g/mL R-Spondin, 5. mu.M A8301, 8. mu.M Y-27632, 12ng/mL Noggin, 12. mu.M SB202190, 1% by volume N2, 1% by volume B27, 10. mu.M MHY1485, 50. mu.g/mL 740Y-P, 2mM HEPES, 5mM Glutamax, 4Mm acetylcysteine, 180U/mL penicillin-streptomycin, 10% by volume FBS.
Example 1
S1, diluting normal tissue cells beside the pleomorphic rhabdomyosarcoma cancer with a culture medium, counting, planting 1000 cells per well and 15 mu L of matrigel thawed at 4 ℃ in advance in a 96-well plate, and placing in a 37 ℃ incubator for first culture for 30min to obtain a normal tissue collagen layer beside the cancer;
s2, carrying out heavy suspension treatment on the rhabdomyosarcoma carcinoma cells by using 15 mu L of culture medium, mixing the heavy suspension treatment mixture with 0.8 mu g of laminin and 15 mu L of matrigel to obtain a first mixture, dripping the first mixture onto a normal tissue collagen layer beside the carcinoma, and carrying out second culture at 37 ℃ for 30min to obtain a double-layer matrix culture system; the concentration of rhabdomyosarcoma carcinoma cells in the resuspension treatment mixture was 35/μ L;
s3, adding 100 mu L of culture medium into a double-layer matrix culture system, performing amplification culture at 37 ℃, performing half liquid change of the culture medium on the 3 rd day of culture, then replacing the culture medium every 2-3 days until 20-40 polymorphic rhabdomyosarcoma organs with the diameter of 200-300 mu M are contained in each hole, adding 2-3 times of cell digestive juice Tryple Express into each hole, and performing cell digestion treatment at 37 ℃ for 3-5min to obtain a second mixture; cell digestion was stopped with DMEM at a FBS concentration of 10 vol% when organoids in the second mixture were dispersed into small clumps and separated from matrigel. After centrifugation, the cells were washed with PBS and harvested, and organoid plating or cryopreservation was performed according to steps S1-S3.
Example 2
A pleomorphic rhabdomyosarcoma organoid was cultured in the same manner as in example 1 except that the content of 740Y-P and MHY1485 in the medium used in this example was different from that of the medium used in example 1, the content of 740Y-P in the medium used in this example was 25. mu.g/mL, and the content of MHY1485 was 5. mu.M.
Comparative example 1
A polymorphic rhabdomyosarcoma organoid was cultured in the same manner as in example 1, except that 740Y-P was not included in the medium used in this example, and the other components and their corresponding contents were the same as those in the medium used in example 1.
Comparative example 2
A polymorphic rhabdomyosarcoma organoid was cultured in the same manner as in example 1, except that MHY1485 was not contained in the medium used in this example, and the other components and their corresponding contents were the same as those in the medium used in example 1.
Comparative example 3
a. Resuspending the rhabdomyosarcoma carcinoma cells with 15 μ L of culture medium, mixing the resuspension mixture with 0.8 μ g of laminin and 15 μ L of matrigel to obtain a first mixture, dripping the first mixture onto a 96-well plate, and culturing at 37 ℃ for 30min to obtain a tumor cell culture system; the concentration of rhabdomyosarcoma carcinoma cells in the resuspension treatment mixture was 35/μ L;
b. adding 100 mu L of culture medium into a tumor culture system, performing amplification culture at 37 ℃, performing half-time medium change on the 3 rd day of culture, then replacing the culture medium every 2-3 days until each hole has 20-40 polymorphic rhabdomyosarcoma organs with the diameter of 200-300 mu M, adding 2-3 times of cell digestive juice Tryple Express into each hole, and performing cell digestive treatment at 37 ℃ for 3-5min to obtain a second mixture; cell digestion was stopped with DMEM at 10% FBS concentration when organoids in the second mixture were dispersed into small clumps and separated from matrigel. After centrifugation, the cells were washed with PBS and harvested, and organoid plating or cryopreservation was performed according to steps S1-S3.
Comparative example 4
A rhabdomyosarcoma organopleioid of pleomorphy was cultured in the same manner as in example 1, except that 740. mu.g/mL of Y-P was contained in the medium used in this example.
Comparative example 5
A polymorphic rhabdomyosarcoma organoid was cultured in the same manner as in example 1, except that the content of MHY1485 in the medium used in this example was 20. mu.M.
Example of detection
1. Polymorphic rhabdomyosarcoma formation and proliferation rate
After 5 years, 34 samples meeting the ethical requirements are collected, a polymorphic rhabdomyosarcoma model is constructed according to the methods of the examples and the comparative examples respectively, the culture is carried out till the 14 th day, the number of organoid single-hole clones with the diameter of 0.2-0.3 mu M is counted under an inverted microscope, the average number of organoid formation of the single sample = the number of total sample clones/34 samples, and the statistical results are shown in table 1.
TABLE 1 polymorphic rhabdomyosarcoma organoid clonogenic status
| Average number of organoids formed in a single sample (× 1000/well) | |
| Example 1 | 1.88 |
| Example 2 | 1.27 |
| Comparative example 1 | 0.55 |
| Comparative example 2 | 0.47 |
| Comparative example 3 | 0.35 |
| Comparative example 4 | 1.04 |
| Comparative example 5 | 0.96 |
As can be seen from table 1, when the samples were obtained from the same sources and cultured for the 14 th day, the number of the rhabdomyosarcoma organoids formed was example 1> example 2> comparative example 4> comparative example 5> comparative example 1> comparative example 2> comparative example 3, which demonstrated that the method of the present disclosure effectively promoted the formation and proliferation of the rhabdomyosarcoma organoid clones and the number of the cultured tumor organoids was large.
2. Polymorphic rhabdomyosarcoma organoid banking success rate
The size of tumor organoid reaches the standard within 1 month, and the number of cryopreserved organoids reaches 1 multiplied by 106The cell is a standard for establishing a library, and the result of counting the establishing success rate of the polymorphic rhabdomyosarcoma organoid library (success rate = success rate/34 × 100%) is shown in table 2.
TABLE 2 statistics table for the database construction of rhabdomyosarcoma organs
| Method | Successful case number (case) of organoid library construction | Organoid construction success rate (%) |
| Example 1 | 30 | 88.24 |
| Example 2 | 27 | 79.41 |
| Comparative example 1 | 17 | 50.00 |
| Comparative example 2 | 16 | 47.06 |
| Comparative example 3 | 13 | 38.24 |
| Comparative example 4 | 23 | 67.65 |
| Comparative example 5 | 22 | 64.71 |
As can be seen from table 2, the results of the studies on the organ-building success of rhabdomyosarcoma of multiforme processed by different methods are respectively example 1> example 2> comparative example 4> comparative example 5> comparative example 1> comparative example 2> comparative example 3, which shows that the method of the present disclosure can effectively perform the in vitro modeling of the rhabdomyosarcoma organoid model of multiforme, and the survival rate of the tumor organoid model is high.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (10)
1. A culture medium for culturing a rhabdomyosarcoma organoid of multiforme, the culture medium contains 740Y-P of 25-75 mu g/mL, MHY1485 of 5-15 mu M, mitotic growth factor of 80-120ng/mL, Wnt agonist of 90-110ng/mL, ROCK inhibitor of 4-15 mu M, BMP inhibitor of 10-15ng/mL, P38 kinase inhibitor of 10-15 mu M, N2 of 0.5-5 vol% based on the total volume of the culture medium, B27 of 0.5-5 vol% based on the total volume of the culture medium, HEPES of 1-5mM, Glutamax of 2-5mM, acetylcysteine of 2-5mM, penicillin-streptomycin of 150-200U/mL and FBS of 5-15 vol% based on the total volume of the culture medium.
2. The medium according to claim 1, wherein, the culture medium contains 30-70 mu g/mL 740Y-P, 6-13 mu M MHY1485, 90-110ng/mL mitotic growth factor, 92-105ng/mL Wnt agonist, 5-14 mu M ROCK inhibitor, 12-14ng/mL BMP inhibitor, 11-14 mu M P38 kinase inhibitor, 1-4 vol% N2 based on the total volume of the culture medium, 1-2 vol% B27 based on the total volume of the culture medium, 2-4mM HEPES, 3-5mM Glutamax, 3-4mM acetylcysteine, 170-190U/mL penicillin-streptomycin and 8-12 vol% FBS based on the total volume of the culture medium.
3. The culture medium of claim 1, wherein the mitotic growth factor is EGF and/or FGF;
the Wnt agonist is selected from one or more of Wnt, Wnt-3a and R-Spondin;
the ROCK inhibitor is A8301 and/or Y-27632;
the BMP inhibitor is selected from Noggin and/or LDN-212854;
the p38 kinase inhibitor is selected from one or more of SB202190, Dilmapimod and SKF-86002.
4. A method of culturing a rhabdomyosarcoma organoid of multiforme, the method comprising:
s1, mixing normal tissue cells beside the pleomorphic rhabdomyosarcoma cancer, a culture medium and matrigel, adding the mixture into the holes of the culture plate, and carrying out first culture to obtain a collagen layer of the normal tissue beside the cancer;
s2, mixing the pleomorphic rhabdomyosarcoma cells, the matrigel and the laminin, then coating the mixture on the collagen layer of the paracancer normal tissue, and carrying out second culture to obtain a double-layer matrix culture system;
s3, adding the culture medium of any one of claims 1-3 into the double-layer matrix culture system for amplification culture.
5. The method as claimed in claim 4, wherein in step S1, the number of normal tissue cells beside the rhabdomyosarcoma carcinoma with polymorphism in each well of the culture plate is 500-1500;
the dosage of the matrigel is 10-15 mu L relative to 10 mu L of the culture medium;
the conditions of the first culture include: the temperature is 30-40 ℃ and the time is 0.2-2 h.
6. The method according to claim 4, wherein in step S2, the step of mixing the rhabdomyosarcoma carcinoma cells, matrigel and laminin together and coating the mixture on the collagen layer of the paracarcinoma normal tissue and performing a second culture comprises:
resuspending the rhabdomyosarcoma carcinoma cell with the culture medium, and mixing a resuspension treatment mixture, the matrigel and the laminin to obtain a first mixture;
dripping the first mixture onto the collagen layer of the paracancer normal tissue, and performing the second culture at 30-40 deg.C for 0.2-2 h.
7. The method according to claim 4, wherein in step S3, the step of adding the culture medium according to any one of claims 1-3 to the double-layer matrix culture system for amplification culture comprises:
adding 50-150 μ L of the culture medium to the two-layer matrix culture system, and performing the amplification culture at 30-40 ℃ until each well has 20-40 polymorphic rhabdomyosarcoma organoids with a diameter of 200-300 μ M.
8. The method of claim 4 or 7, further comprising: mixing the amplification culture product obtained by the amplification culture in the step S3 with a digestive juice, and performing cell digestion treatment to obtain a second mixture;
the second mixture is allowed to terminate cell digestion, and the cells obtained after termination of cell digestion are subcultured.
9. A rhabdomyosarcoma organoid of pleomorphism obtained by culturing according to the method of any one of claims 4 to 8.
10. Use of the rhabdomyosarcoma organoid of polymorphic form of claim 9 in drug screening and tumor mechanism research.
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